Genetic Studies Of Rotavirus Pathogenesis And Development Of Rotavirus Vaccines

先前，在涉及非生新生猪与猪轮状病毒毒株（SB-1A）和人轮状病毒毒株（DS-1）及其重组体的半同源系统的研究中，我们证明：(i)猪轮状病毒第3 （VP3）、第4 （VP4）、第9 （VP7）和第10 （NSP4）基因在仔猪轮状病毒感染的毒力中各自发挥重要的独立作用；（ii）所有四种猪轮状病毒毒力相关基因都是诱导仔猪腹泻和排出轮状病毒所必需的。这些观察结果提示了一种潜在的新策略，用于衰减具有重要流行病学意义的野生型人轮状病毒，并将其应用于开发安全有效的疫苗。此前，我们开发了基于恒河猴(RRV)-，牛(UK)-和猪（Gottfried）的多价候选疫苗，旨在为具有流行病学重要性的VP7 (G)血清型1- 4,8和9提供抗原覆盖。今年，为了研究小鼠轮状病毒EB株的哪些基因参与了同源小鼠模型中腹泻的诱导，我们在4-5日龄的CD-1小鼠中连续传代了一种细胞培养适应的无毒（非致泻性）小鼠轮状病毒EB株，直到病毒变得有毒性（致泻性）。然后将该毒力病毒接种于原代非洲绿猴肾（AGMK）细胞上，连续传代18次。第18个细胞培养传代病毒在小鼠幼崽中显示为无毒，随后在CD-1小鼠中连续传代时再次具有毒性。该毒力病毒在原代AGMK细胞中连续传代18次后，在小鼠中变得无毒。对本研究中产生的毒株和无毒性病毒的11个基因进行测序，结果显示，与无毒性病毒相比，有毒性病毒在基因4（编码外衣壳VP4）和基因10（编码病毒肠毒素NSP4）上有明显的突变，而在其他基因上没有明显的突变。此外，病毒在小鼠体内的复制效率在毒性病毒和无毒性病毒之间没有显著差异。我们制备了3个杆状病毒重组体，表达NSP4蛋白，这些杆状病毒重组体分别来自小鼠高传代强毒EB病毒（样本A）、小鼠低传代强毒EB病毒（样本B）和细胞培养传代强毒EB病毒（样本C）。经口腔接种后，A组91.6%（11 / 12）小鼠出现腹泻；B组12.5% (1 / 8)；C组为0%(0 / 12)，说明NSP4蛋白在该模型的发病机制中发挥了重要作用。

Previously, in studies involving a semi-homologous system of gnotobiotic newborn pigs and a virulent porcine rotavirus strain (SB-1A) and an avirulent human rotavirus strain (DS-1) and their reassortants, we demonstrated that: (i) the third (VP3), fourth (VP4), ninth (VP7), and tenth (NSP4) porcine rotavirus gene each play an important independent role in the virulence of rotavirus infection in piglets; and (ii) all four of the porcine rotavirus virulence-associated genes are required for the induction of diarrhea and the shedding of rotavirus by piglets. These observations suggested a potential new strategy for attenuation of wild-type human rotaviruses of major epidemiologic importance and its application to the development of a safe and effective vaccine. Previously, we developed rhesus (RRV)-, bovine (UK)-, and porcine (Gottfried)-based multivalent vaccine candidates which were designed to provide antigenic coverage for VP7 (G) serotypes 1-4, 8 and 9 of epidemiologic importance. This year, in order to study which gene(s) of a murine rotavirus EB strain are involved in induction of diarrhea in a homologous mouse model, we passaged a cell culture-adapted avirulent (non-diarrheagenic) murine rotavirus EB strain serially in 4-5 day-old CD-1 mice until the virus became virulent (diarrheagenic). This virulent virus was then inoculated onto primary African green monkey kidney (AGMK) cells and passaged serially 18 times. The 18th cell culture-passaged virus that was shown to be avirulent in mouse pups became virulent again during subsequent serial passage in CD-1 mice. This virulent virus when passaged again serially 18 times in primary AGMK cells became avirulent in mice. Sequencing of all 11 genes of selected virulent and avirulent viruses generated in this study, revealed that virulent viruses when compared with avirulent viruses bore (i) distinct mutations in gene 4 (encoding outer capsid VP4) and in gene 10 (encoding viral enterotoxin NSP4) and (ii) no significant mutations in other genes. In addition, no significant difference in virus replication efficiency in mice was observed between virulent and avirulent viruses. We generated 3 baculovirus recombinants expressing the NSP4 protein derived from high mouse-passaged virulent EB virus (sample A), low mouse-passaged virulent EB virus (sample B), or cell culture-passaged avirulent EB virus (sample C). Upon oral inoculation in mouse pups, the development of diarrhea was observed in 91.6% (11 of 12) in sample A group; 12.5% (1 of 8) in sample B group; and 0% (0 of 12) in sample C group, indicating that the NSP4 protein played an important role in pathogenesis in this model.